Amyloid-Beta Modulates Low-Threshold Activated Voltage-Gated L-Type Calcium Channels of Arcuate Neuropeptide Y Neurons Leading to Calcium Dysregulation and Hypothalamic Dysfunction

Abstract

Weight loss is an early manifestation of Alzheimer's disease that can precede the cognitive decline, raising the possibility that amyloid-β (Aβ) disrupts hypothalamic neurons critical for the regulation of body weight. We previously reported that, in young transgenic mice overexpressing mutated amyloid precursor protein (Tg2576), Aβ causes dysfunction in neuropeptide Y (NPY)-expressing hypothalamic arcuate neurons before plaque formation. In this study, we examined whether Aβ causes arcuate NPY neuronal dysfunction by disrupting intracellular Ca²⁺ homeostasis. Here, we found that the L-type Ca²⁺ channel blocker nimodipine could hyperpolarize the membrane potential, decrease the spontaneous activity, and reduce the intracellular Ca²⁺ levels in arcuate NPY neurons from Tg2576 brain slices. In these neurons, there was a shift from high to low voltage-threshold activated L-type Ca²⁺ currents, resulting in increased Ca²⁺ influx closer to the resting membrane potential, an effect recapitulated by Aβ_1-42 and reversed by nimodipine. These low voltage-threshold activated L-type Ca²⁺ currents were dependent in part on calcium/calmodulin-dependent protein kinase II and IP₃ pathways. Furthermore, the effects on intracellular Ca²⁺ signaling by both a positive (ghrelin) and negative (leptin) modulator were blunted in these neurons. Nimodipine pretreatment restored the response to ghrelin-mediated feeding in young (3-5 months), but not older (10 months), female Tg2576 mice, suggesting that intracellular Ca²⁺ dysregulation is only reversible early in Aβ pathology. Collectively, these findings provide evidence for a key role for low-threshold activated voltage gated L-type Ca²⁺ channels in Aβ-mediated neuronal dysfunction and in the regulation of body weight.SIGNIFICANCE STATEMENT Weight loss is one of the earliest manifestations of Alzheimer's disease (AD), but the underlying cellular mechanisms remain unknown. Disruption of intracellular Ca²⁺ homeostasis by amyloid-β is hypothesized to be critical for the early neuronal dysfunction driving AD pathogenesis. Here, we demonstrate that amyloid-β causes a shift from high to low voltage-threshold activated L-type Ca²⁺ currents in arcuate neuropeptide Y neurons. This leads to increased Ca²⁺ influx closer to the resting membrane potential, resulting in intracellular Ca²⁺ dyshomeostasis and neuronal dysfunction, an effect reversible by the L-type Ca²⁺ channel blocker nimodipine early in amyloid-β pathology. These findings highlight a novel mechanism of amyloid-β-mediated neuronal dysfunction through L-type Ca²⁺ channels and the importance of these channels in the regulation of body weight.

Keywords: Alzheimer's disease; electrophysiology; ghrelin; hypothalamus; leptin; neuropeptide Y.

Figure 1.

The membrane potential and spike frequency in arcuate NPY neurons from Tg2576 mice are sensitive to the L-type Ca²⁺ channel blocker nimodipine (NMD). A, Brain slices containing the hypothalamic arcuate nucleus from young (3- to 4-month-old) NPY-GFP mice with APP_swe transgene (Tg2576) were used for whole-cell patch-clamp recordings. Representative traces are shown before and after nimodipine (2 μm). B, Application of the L-type voltage-gated Ca²⁺ channel antagonist nimodipine (2 μm) hyperpolarized the membrane potential of arcuate NPY neurons in Tg2576 brain slices. n = 8 cells from 6 mice per group. Statistical analysis: two-tailed paired Student's t test (t = 4.508, df = 7, p = 0.0028). C, Application of the L-type voltage-gated Ca²⁺ channel antagonist nimodipine (2 μm) reduced the spike frequency of arcuate NPY neurons in Tg2576 brain slices. n = 8 cells from 6 mice per group. Statistical analysis: two-tailed paired Student's t test (t = 4.548, df = 7, p = 0.0026). D, Application of the N-type (ω-Cgtx-GVIA,1 μm) or the P/Q-type Ca²⁺ channel (ω-AgaIVA, 1 μm) antagonists had no effect on membrane potential of arcuate NPY neurons in Tg2576 brain slices. n = 4 cells from 3 mice per group. Statistical analysis: repeated-measures one-way ANOVA (F_{(1.030, 3.089)} = 0.968, p = 0.3993). E, Application of the N-type (ω-Cgtx-GVIA,1 μm) or the P/Q-type Ca²⁺ channel (ω-AgaIVA, 1 μm) antagonists had no effect on the spike frequency of arcuate NPY neurons in Tg2576 brain slices. n = 4 cells from 3 mice per group. Statistical analysis: repeated-measures one-way ANOVA (F_{(1.947, 5.841)} = 1.168, p = 0.3727). Data are mean ± SEM. **p < 0.01.

Figure 2.

Arcuate NPY neurons from Tg2576 brain slices or exogenous Aβ_1–42-treated WT brain slices have increased cytoplasmic-free Ca²⁺ levels that can be reversed by the L-type Ca²⁺ channel blocker nimodipine (NMD). A, Arcuate NPY neurons from Tg2576 mice have increased cytoplasmic-free Ca²⁺ levels compared with WT mice that are decreased by nimodipine. Cytoplasmic-free Ca²⁺ was measured using fura-2 AM in arcuate NPY neurons isolated by enzymatic digestion from WT or Tg2576 mice. After obtaining baseline levels, cells were perfused with nimodipine (2 μm). n = 18–21 cells from ≥ 3 mice per group. Statistical analysis: one-way ANOVA (F_(3,74) = 9.738, p < 0.0001, followed by post hoc Tukey's multiple comparisons). **p < 0.01. B, Exogenous Aβ_1–42-treated arcuate NPY neurons from WT mice have increased cytoplasmic-free Ca²⁺ levels that are decreased by nimodipine. Cytoplasmic-free Ca²⁺ was measured using fura-2 AM in arcuate NPY neurons isolated by enzymatic digestion from WT mice. After obtaining baseline levels, cells were first perfused with oligomeric Aβ_1–42 (100 or 300 nm) followed by nimodipine (2 μm). n = 4 or 5 cells from 2 mice per group. Statistical analysis: one-way ANOVA (F_(3,14) = 5.028, p = 0.0143, followed by post hoc Tukey's multiple comparisons). *p < 0.05. Data are mean ± SEM.

Figure 3.

Arcuate NPY neurons from Tg2576 brain slices or exogenous Aβ_1–42-treated WT brain slices have a left shift in the peak L-type Ca²⁺ current I–V curve that can be reversed by the L-type Ca²⁺ channel blocker nimodipine (NMD). A, L-type Ca²⁺ currents in arcuate NPY neurons from Tg2576 brain slices show a propensity for channel opening close to the resting potential compared with arcuate NPY neurons from WT brain slices. Representative traces are shown using the holding potential −60 mV to different stepped potentials from −50 to 20 mV (showing −30 to 10 mV). B, I–V relationships for L-type Ca²⁺ currents in arcuate NPY neurons from WT or Tg2576 brain slices. There was a left shift in the peak L-type Ca²⁺ current I–V curve in arcuate NPY neurons from Tg2576 brain slices compared with WT brain slices. The peak L-type Ca²⁺ current in the I–V curve occurred at −20 mV for arcuate NPY neurons from Tg2576 brain slices compared with 0 mV for arcuate NPY neurons from WT slices. At −20 mV, arcuate NPY neurons from Tg2576 brain slices have significantly higher L-type Ca²⁺ currents compared with arcuate NPY neurons from WT brain slices. Data are represented as L-type Ca²⁺ current density (pA/pF). n = 19–27 per group from ≥ 12 mice per group. Statistical analysis: two-tailed unpaired Student's t test (t = 2.832, df = 45, p = 0.0069). C, The peak L-type Ca²⁺ current I–V curve in arcuate NPY neurons from WT brain slices treated with oligomeric Aβ_1–42 (100 nm) recapitulates the left shift seen in arcuate NPY neurons from Tg2576 brain slices. The peak L-type Ca²⁺ current in the I–V curve occurred between −10 mV and −20 mV for arcuate NPY neurons after application of oligomeric Aβ_1–42 (100 nm) slices compared with 0 mV at baseline (vehicle treatment). At −20 mV, arcuate NPY neurons from oligomeric Aβ_1–42 (100 nm)-treated WT brain slices have significantly higher L-type Ca²⁺ currents compared with arcuate NPY neurons from vehicle-treated WT brain slices. Data are represented as L-type Ca²⁺ current density (pA/pF). n = 6 per group from ≥ 4 mice per group. Statistical analysis: two-tailed paired Student's t test (t = 4.598, df = 5, p = 0.0059). D, L-type Ca²⁺ current induced at −10 mV were significantly decreased after application of nimodipine (2 μm) in arcuate NPY neurons from WT brain slices, Tg2576 brain slices, and WT brain slices treated with oligomeric Aβ_1–42 (100 nm). n = 5–8 per group from ≥ 4 mice per group. Statistical analysis: two-tailed paired Student's t test (WT: t = 9.806, df = 7, p < 0.0001; Tg2576: t = 4.694, df = 5, p = 0.0054; Aβ_1–42: t = 2.912, df = 4, p = 0.0436). Data are mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001.

Figure 4.

Pharmacologic inhibition of CaMKII or IP₃ can partially reverse the left shift in the peak L-type Ca²⁺ current I–V curve in arcuate NPY neurons from Tg2576 brain slices. A, I–V relationships for L-type Ca²⁺ currents in arcuate NPY neurons from Tg2576 brain slices after application of the CaMKII antagonist KN93 (10 μm). The left-shifted I–V curve was partially reversed after application of KN93. The peak L-type Ca²⁺ current in the I–V curve occurred at −20 mV for arcuate NPY neurons from vehicle-treated Tg2576 brain slices compared with −10 mV after KN93 application. At −20 mV, arcuate NPY neurons from KN93 (10 μm)-treated Tg2576 brain slices have significantly lower L-type Ca²⁺ currents compared with vehicle-treated Tg2576 brain slices. Data are represented as L-type Ca²⁺ current density (pA/pF). n = 5 per group from 4 mice per group. Statistical analysis: two-tailed paired Student's t test (t = 2.854, df = 4, p = 0.0462). B, I–V relationships for L-type Ca²⁺ currents in arcuate NPY neurons from Tg2576 brain slices after application of the IP₃ antagonist 2APB (50 μm). The left-shifted I–V curve was partially reversed after application of 2APB. The peak L-type Ca²⁺ current in the I–V curve occurred at −20 mV for arcuate NPY neurons from Tg2576 brain slices compared with −10 mV after 2APB application. At −20 mV, arcuate NPY neurons from 2APB (50 μm)-treated Tg2576 brain slices have significantly lower L-type Ca²⁺ currents compared with vehicle-treated Tg2576 brain slices. Data are represented as L-type Ca²⁺ current density (pA/pF). n = 4 per group from 3 mice per group. Statistical analysis: two-tailed paired Student's t test (t = 3.656, df = 3, p = 0.0353). C, I–V relationships for L-type Ca²⁺ currents in arcuate NPY neurons from Tg2576 brain slices after application of the phospholipase C antagonist U733122 (10 μm). The left-shifted I–V curve was unchanged after application of U733122. The peak L-type Ca²⁺ current in the I–V curve occurred at −20 mV for arcuate NPY neurons from Tg2576 brain slices before and after U733122 application. At −20 mV, arcuate NPY neurons from U733122 (10 μm)-treated Tg2576 brain slices have similar L-type Ca²⁺ currents compared with vehicle-treated Tg2576 brain slices. n = 5 cells per group from 3 mice per group. Statistical analysis: two-tailed paired Student's t test (t = 0.3566, df = 4, p = 0.7394). Data are mean ± SEM.

Figure 5.

Ghrelin and leptin fail to modulate cytoplasmic-free Ca²⁺ levels in arcuate NPY neurons from Tg2576 mice. A, Ghrelin increases cytoplasmic-free Ca²⁺ levels in arcuate NPY neurons from WT but not Tg2576 mice. Cytoplasmic-free Ca²⁺ was measured using fura-2 AM in arcuate NPY neurons isolated by enzymatic digestion from WT or Tg2576 mice. After obtaining baseline levels, cells were perfused with ghrelin (100 nm). n = 34 WT and n = 7 Tg2576 cells from ≥ 3 mice per group. Statistical analysis: two-tailed paired Student's t test (WT: t = 2.664, df = 34, p = 0.0117; Tg2576: t = 1.020, df = 7, p = 0.3471). B, Leptin decreases cytoplasmic-free Ca²⁺ levels in arcuate NPY neurons from WT but not Tg2576 mice. Cytoplasmic-free Ca²⁺ was measured using fura-2 AM in arcuate NPY neurons isolated by enzymatic digestion from WT or Tg2576 mice. After obtaining baseline levels, cells were perfused with leptin (100 nm). n = 25 WT and n = 20 Tg2576 cells from ≥ 3 mice per group. Statistical analysis: two-tailed paired Student's t test (WT: t = 3.032, df = 24, p = 0.0057; Tg2576: t = 2.030, df = 19, p = 0.0566). C, I–V relationships for L-type Ca²⁺ currents in arcuate NPY neurons from WT brain slices after application of leptin (100 nm). Compared with vehicle treatment, leptin significantly decreased the peak L-type Ca²⁺ current at 0 mV. Data are represented as L-type Ca²⁺ current density (pA/pF). n = 7 cells per group. Statistical analysis at 0 mV: two-tailed paired Student's t test (t = 3.601, df = 6, p = 0.0114). D, I–V relationships for L-type Ca²⁺ currents in arcuate NPY neurons from Tg2576 brain slices after application of leptin (100 nm). Compared with vehicle treatment, there is no significant difference in L-type Ca²⁺ currents. Data are represented as L-type Ca²⁺ current density (pA/pF). n = 4 cells per group. Statistical analysis at −20 mV: two-tailed paired Student's t test (t = 1.379, df = 3, p = 0.2617). Data are mean ± SEM. *p < 0.05. **p < 0.01.

Figure 6.

Nimodipine can restore the neurophysiological responses to ghrelin in arcuate NPY neurons treated with oligomeric Aβ_1–42. A, B, Brain slices containing hypothalamic arcuate nucleus from young (3- to 4-month-old) NPY-GFP mice were used for whole-cell patch-clamp recordings. Application of ghrelin (100 nm) depolarized the membrane potential and increased spike frequency of arcuate NPY neurons. Exogenous oliogmeric Aβ_1–42 (100 nm) depolarized the membrane potential of arcuate NPY neurons and inhibited its response to ghrelin (100 nm). Nimodipine (NMD, 2 μm) restored the ghrelin-mediated depolarization of arcuate NPY neurons treated with exogenous oligomeric Aβ_1–42. Statistical analysis: two-tailed paired Student test for comparison of vehicle to ghrelin-treated arcuate NPY neurons (membrane potential: t = 6.444, df = 15, p < 0.001, n = 16 cells from 13 mice per group; spike frequency: t = 3.742, df = 13, p = 0.0025, n = 14 cells from 12 mice per group). Repeated-measures one-way ANOVA comparing vehicle, Aβ_1–42, and ghrelin treatments (membrane potential: F_{(1.579, 12.63)} = 7.577, p = 0.0096, n = 9 cells from 5 mice per group; spike frequency: F_{(1.365, 9.557)} = 3.941, p = 0.0679, n = 8 cells from 5 mice per group), followed by post hoc Tukey's multiple comparisons. Repeated-measures one-way ANOVA comparing vehicle, Aβ_1–42, nimodipine, and ghrelin treatments (membrane potential: F_{(1.883, 11.30)} = 8.785, p = 0.0014, n = 7 cells from 4 mice per group; spike frequency: F_{(2.125, 12.75)} = 3.220, p = 0.0716, n = 7 cells from 4 mice per group) followed by post hoc Tukey's multiple comparisons. *p < 0.05. **p < 0.01. ***p < 0.001. Data are mean ± SEM. C, Nimodipine restored the ghrelin-mediated increase in cytoplasmic-free Ca²⁺ levels in arcuate NPY neurons treated with exogenous oligomeric Aβ_1–42. Cytoplasmic-free Ca²⁺ was measured using fura-2 AM in arcuate NPY neurons isolated by enzymatic digestion from young (3- to 4-month-old) WT NPY-GFP mice. After obtaining baseline levels, cells were perfused sequentially with oligomeric Aβ_1–42 (100 nm), nimodipine (2 μm), and then ghrelin (100 nm). Statistical analysis: repeated-measures one-way ANOVA comparing vehicle, Aβ_1–42, nimodipine, and ghrelin treatments (F_{(2.128, 44.69)} = 13.90, p < 0.0001, n = 22 cells from 4 mice) followed by post hoc Tukey's multiple comparisons. *p < 0.05. **p < 0.01. ***p < 0.001. Data are mean ± SEM.

Figure 7.

Pretreatment with nimodipine can restore ghrelin-mediated feeding behavior in young (3–5 months), but not older (10 months), Tg2576 mice. A, Ghrelin (0.5 mg per kg body weight, i.p.) increased feeding in young (3–5 months) WT but not Tg2576 mice. n = 9 WT and n = 14 Tg2576 mice. Statistical analysis: two-tailed paired Student's t test (WT: t = 2.337, df = 8, p = 0.0476; Tg2576: t = 1.034, df = 13, p = 0.3201). B, Nimodipine (10 mg per kg body weight, i.p.) administered 1 h before ghrelin (0.5 mg per kg body weight, i.p.) restored the ghrelin-mediated feeding behavior in young (3–5 months) Tg2576 mice. n = 12 WT and n = 12 Tg2576 mice. Statistical analysis: two-tailed paired Student's t test (WT: t = 3.986, df = 11, p = 0.0021; Tg2576: t = 4.284, df = 11, p = 0.0013). C, Nimodipine (10 mg per kg body weight, i.p.) administered 1 h before ghrelin (0.5 mg per kg body weight, i.p.) failed to restore the ghrelin-mediated feeding behavior in older (10 months) Tg2576 mice. n = 12 WT and n = 14 Tg2576 mice. Statistical analysis: two-tailed paired Student's t test (WT: t = 4.128, df = 11, p = 0.0017; Tg2576: t = 1.754, df = 13, p = 0.103). Data are expressed as individual mice. *p < 0.05; **p < 0.01; paired t test. n.s. = not significant.

Figure 8.

There are no significant differences in the L-type Ca²⁺ currents in hypothalamic paraventricular neurons from Tg2576 or WT mice. I–V curves for L-type Ca²⁺ currents in randomly selected hypothalamic paraventricular non-NPY neurons from WT or Tg2576 slices. There are no significant differences between hypothalamic paraventricular non-NPY neurons from WT and Tg2576 slices. The peak L-type Ca²⁺ current occurred at −10 mV for hypothalamic paraventricular neurons in both WT and Tg2576 brain slices with no significant differences in the amplitudes. Statistical analysis: two-tailed unpaired Student's t test (t = 0.2289, df = 6, p = 0.8266). Data are represented as L-type Ca²⁺ current density (pA/pF). n = 4 cells per group from ≥ 3 mice per group. Data are mean ± SEM.